Sensitive immunochromatographic assay

ABSTRACT

Methods for quantitatively measuring the amount of an analyte of interest in a fluid sample, and kits useful in the methods, are disclosed. The methods involve providing a solid phase apparatus comprising a membrane having an application point, a sample capture zone, and a control capture zone, where the sample capture region is between the application point and the control capture zone; and providing a sample collection apparatus comprising a population of analyte binding particles or a population of analyte coated particles. In the assays, a fluid sample is introduced into the sample collection apparatus, and the resultant mixture is applied to the application point of the membrane. The fluid allows transport components of the assay by capillary action to and through the sample capture zone and subsequently to and through the control capture zone. The amount of analyte in the fluid sample is related (e.g., either directly or inversely) to a corrected particle amount, which can be determined, for example, as a ratio of the amount of particles in the sample capture zone and the amount of particles in the control capture zone.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/162,138, filed Jun. 3, 2002, which is a continuation-in-part of U.S.application Ser. No. 10/120,774, filed Apr. 10, 2002, the entireteachings which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Quantitative analysis of cells and analytes in fluid samples,particularly bodily fluid samples, often provides critical diagnosticand treatment information for physicians and patients. Quantitativeimmunoassays utilize the specificity of the antigen (Ag)-antibody (Ab)reaction to detect and quantitate the amount of an Ag or Ab in a sample.In solid phase immunoassays, one reagent (e.g., the Ag or Ab) isattached to a solid surface, facilitating separation of bound reagentsor analytes from free reagents or analytes. The solid phase is exposedto a sample containing the analyte, which binds to its Ag or Ab; theextent of this binding is quantitated to provide a measure of theanalyte concentration in the sample. Transduction of the binding eventinto a measurable signal, however, is affected by a number oflimitations, including constraints of particle movement on the solidphase, which affect the specificity and applicability of quantitativeimmunoassays.

SUMMARY OF THE INVENTION

The invention relates to methods of measuring the amount of an analyteof interest in a fluid sample, using a solid phase assay (e.g., asandwich immunoassay or an inhibition immunoassay), in which an analyteof interest and a capture reagent are used as part of a specific bindingpair; and to kits for use in the methods.

In the methods of the invention, a solid phase apparatus is provided,which includes a membrane strip having an application point, a samplecapture zone and a control capture zone; the sample capture zone isbetween the application point and the control capture zone. A samplecapture reagent (e.g., an agent that binds to the analyte of interest,such as an antibody to the analyte of interest) is immobilized in thesample capture zone. A control capture reagent (e.g., an agent thatbinds to the analyte binding particles, such as an anti-immunoglobulinantibody) is immobilized in the control capture zone. Also provided is asample collection apparatus containing a population of particles, suchas liposomes, colloidal gold, or organic polymer latex particles, storedin a stable form.

In “sandwich” immunoassays of the invention, the particles are “analytebinding” particles that are coated with a binding agent (e.g., anantibody) to the analyte of interest. In “competitive” or “inhibition”assays, the particles are “analyte coated” particles that are coatedwith analyte of interest. In either type of assay, the particles can belabeled, using a colorimetric, fluorescent, luminescent,chemiluminescent, or other appropriate label, to facilitate detection.

In one embodiment of the methods, a fluid sample to be assessed for theanalyte of interest is introduced into the sample collection apparatus,and a buffer is subsequently introduced into the mixed fluid sample. Inanother embodiment of the methods, a buffer is introduced into thesample collection apparatus, and the fluid sample to be assessed for theanalyte of interest is subsequently introduced. In a third embodiment ofthe methods, the fluid sample is formed by introducing a solid into abuffer, and the fluid sample is subsequently introduced into the samplecollection apparatus. In any of these embodiments, a buffered, mixedfluid sample containing the particles is produced.

In a sandwich assay, analyte of interest present in the sample interactswith the analyte binding particles, resulting in contacted analytebinding particles within the mixed fluid sample. The buffered, mixedfluid sample is applied to the application point of the membrane stripof the solid phase apparatus. The solid phase apparatus is thenmaintained under conditions which are sufficient to allow capillaryaction of fluid to transport particles to and through the sample capturezone.

The sample capture reagent interacts with contacted analyte bindingparticles, resulting in arrest of particles in the sample capture zone.Capillary action of the fluid further mobilizes the contacted analytebinding particles not only to and through the sample capture zone, butalso to and through the control capture zone, where they bind to thecontrol capture reagent. Capillary action of the fluid continues tomobilize the remaining unbound particles past the control capture zone(e.g., into a wicking pad). The amount of analyte binding particles thatare arrested in the sample capture zone, and in the control capturezone, are then determined.

The amount of analyte of interest in the fluid sample is thendetermined. For example, the amount of analyte of interest in the fluidsample can be determined as a ratio between 1) the amount of analytebinding particles that are arrested in the sample capture zone, and 2)the amount of analyte binding particles in the control capture zone.Alternatively, the amount of analyte of interest in the fluid sample canbe determined as a ratio between 1) the amount of analyte bindingparticles that are arrested in the sample capture zone, and 2) the sumof the amount of analyte binding particles in the control capture zoneand the amount of analyte binding particles that are arrested in thesample capture zone.

In a competitive or inhibition type of assay, the buffered, mixed fluidsample is applied to the application point of the membrane strip of thesolid phase apparatus. The solid phase apparatus is then maintainedunder conditions which are sufficient to allow capillary action of fluidto transport particles to and through the sample capture zone.

The sample capture reagent interacts with analyte-coated particles;interaction of the sample capture reagent and the analyte-coatedparticles results in arrest of analyte-coated particles in the samplecapture zone. Because of competition between the analyte-coatedparticles and analyte (if present) in the sample for binding sites onthe sample capture reagent in the sample capture zone, the amount ofanalyte-coated particles arrested in the sample capture zone isinversely proportional to the amount of analyte in the sample. Capillaryaction of the fluid further mobilizes the analyte-coated particles notonly to the sample capture zone, but also to the control capture zone,where they bind to the control capture reagent. The amount ofanalyte-coated particles that are arrested in the sample capture zone,and in the control capture zone, are then determined.

The amount of analyte of interest in the fluid sample is thendetermined. For example, the amount of analyte of interest in the fluidsample is inversely related to a ratio between 1) the amount ofanalyte-coated particles that are arrested in the sample capture zone,and 2) the amount of analyte-coated particles in the control capturezone. Alternatively, the amount of analyte of interest in the fluidsample is inversely related to a ratio between 1) the amount ofanalyte-coated particles that are arrested in the sample capture zone,and 2) the sum of the amount of analyte-coated particles in the controlcapture zone and the amount of analyte-coated particles that arearrested in the sample capture zone.

The flow of fluid through a solid phase in such quantitative assayscontributes to the dynamic nature of the assays: the amount of analytebinding to particles, as well as the location of particles in relationto positions on the solid phase, is in flux. The structure of the solidphase reactants, as well as the viscosity of the fluid sample and otherfactors, can thereby contribute to limitations on specificity of theassays. The methods of the invention reduce certain constraints on thedynamic nature of the assays, thereby allowing more accuratedetermination of the amounts of analytes of interest in solutions. Forexample, in the sandwich assays, because the fluid sample to be assayedfor the analyte of interest is mixed with the analyte binding particlesprior to application to the membrane, there is a longer time for theanalyte of interest to bind to the analyte binding particles prior tothe capture reaction which occurs in the membrane. Furthermore, becausethe interaction between the analyte of interest and the analyte bindingparticles occurs in the fluid phase, there is faster and more efficientbinding because of greater mobility of the particles, than there wouldbe in the same interaction between analyte of interest and analytebinding particles in the matrix of the membrane of the solid phaseapparatus. In both the sandwich assays and the inhibition (competitive)assays, it is possible to increase the volume of particles used withoutoverloading the membrane, thereby increasing sensitivity of the assay.In addition, the particles pass over the capture zones in a continuousmanner through the capillary action of the fluid, rather than in a quickwave on the crest of a fluid front, allowing more effective capture ofparticles and thereby enhancing sensitivity of the assays.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to methods of quantitatively measuringthe amount of an analyte using assays, particularly quantitativeimmunochromatographic assays, and kits therefor.

An “assay,” as used herein, refers to an in vitro procedure for analysisof a sample to determine the presence, absence, or quantity of one ormore analytes. The assays of the inventions utilize an analyte and ananalyte binding agent. The analyte and the analyte binding agent aremembers of a specific “binding pair,” in which a first member of thebinding pair (e.g., analyte) reacts specifically with a second member(e.g., the binding agent). One or both members of the binding pair canbe an antibody. For example, a first member of the binding pair (e.g.,an analyte of interest) can be an antibody, and a second member of thebinding pair (e.g., a binding agent) can be anti-immunoglobulinantibody; alternatively, the first member of the binding pair (e.g., theanalyte) can be an antigen, and the second member of the binding pair(e.g., the binding agent) can be an antibody.

In one embodiment, the assay is an “immunoassay” which utilizesantibodies as a component of the procedure. In a preferred embodiment,the immunoassay is a “sandwich” assay, which is a test for an analyte inwhich a fluid sample to be assessed for the presence or absence, orquantity of analyte, is contacted with particles coated with an analytebinding agent, such as antibodies to the analyte, and the resultantmixture is applied to a membrane and subsequently moves by capillaryaction through the membrane. A positive result is indicated by detectionof interaction between analyte and analyte binding agent-coatedparticles in a capture zone of the membrane, the amount of analytebinding agent-coated particles in the capture zone being related to theamount of analyte in the fluid sample. In another preferred embodiment,the immunoassay is an “inhibition” or “competitive” assay, which is atest for an analyte in which a fluid test sample to be assessed for thepresence or absence, or quantity of analyte, is contacted with particlescoated with the analyte, and the resultant mixture is applied to amembrane and subsequently moves by capillary action the system throughthe membrane. A positive result is indicated by detection of interactionbetween analyte binding agent and analyte-coated particles in a capturezone of the membrane, the amount of analyte-coated particles in thecapture zone being inversely related to the amount of analyte in thefluid sample.

In another embodiment of the assays of the invention, neither theanalyte nor the binding agent are antibodies: for example, the firstmember of the binding pair can be a ligand, and the second member of thebinding pair can be a receptor; alternatively, the first member of thebinding pair can be a lectin, and the second member of the binding paircan be a sugar. In still another embodiment, the first member of thebinding pair can be a nucleic acid (e.g., DNA, RNA), and the secondmember of the binding pair can be a nucleic acid which specificallyhybridizes to the first member of the binding pair. “Specifichybridization,” as used herein, refers to the ability of a first nucleicacid to hybridize to a second nucleic acid in a manner such that thefirst nucleic acid does not hybridize to any nucleic acid other than tothe second nucleic acid (e.g., when the first nucleic acid has a highersimilarity to the second nucleic acid than to any other nucleic acid ina sample wherein the hybridization is to be performed). “Stringencyconditions” for hybridization is a term of art which refers to theincubation and wash conditions, e.g., conditions of temperature andbuffer concentration, which permit hybridization of a particular nucleicacid to a second nucleic acid; the first nucleic acid may be perfectly(i.e., 100%) complementary to the second, or the first and second mayshare some degree of complementarity which is less than perfect (e.g.,70%, 75%, 80%, 85%, 90%, 95%). For example, certain high stringencyconditions can be used which distinguish perfectly complementary nucleicacids from those of less complementarity. “High stringency conditions”,“moderate stringency conditions” and “low stringency conditions” fornucleic acid hybridizations are explained on pages 2.10.1-2.10.16 andpages 6.3.1-6.3.6 in Current Protocols in Molecular Biology (Ausubel, F.M. et al., “Current Protocols in Molecular Biology”, John Wiley & Sons,(1998), the entire teachings of which are incorporated by referenceherein). The exact conditions which determine the stringency ofhybridization depend not only on ionic strength (e.g., 0.2×SSC,0.1×SSC), temperature (e.g., room temperature, 42° C., 68° C.) and theconcentration of destabilizing agents such as formamide or denaturingagents such as SDS, but also on factors such as the length of thenucleic acid sequence, base composition, percent mismatch betweenhybridizing sequences and the frequency of occurrence of subsets of thatsequence within other non-identical sequences. Thus, equivalentconditions can be determined by varying one or more of these parameterswhile maintaining a similar degree of identity or similarity between thetwo nucleic acid molecules.

Regardless of the composition of the analyte and the binding agent,these two components nevertheless form a specific binding pair, in whichthe first member reacts specifically with the second member. Specificinteraction between the members of the binding pair indicates that thefirst member of the binding pair preferentially binds or otherwiseinteracts with the second member of the binding pair, preferably to theexclusion of any binding to another compound in the assay.

The terms, “analyte” or “analyte of interest,” as used herein, refer toa first member of a binding pair as described above. The analyte is amolecule or compound for which the amount will be measured. The analytecan be in the form of a solid, such as a dry substance (e.g., a powder,a particulate; spore; or other particle), or can be in the form of afluid (e.g., a solid as described above that has been dissolved orsuspended in a fluid; or other liquid sample). Examples of analytesinclude spores; proteins, such as hormones or enzymes; glycoproteins;peptides; small molecules; polysaccharides; antibodies; nucleic acids;drugs; toxins (e.g., environmental toxins); viruses or virus particles;portions of a cell wall; and other compounds. In a preferred embodiment,the analyte is “immunogenic,” which indicates that antibodies (asdescribed below) can be raised to the analyte, or to an analyte that isbound to a carrier (e.g., a hapten-carrier conjugate, for whichantibodies can be raised to the hapten). In some representativeembodiments, the analyte of interest can be myoglobin; CK-MB; troponinI; PSA; digoxin; theophylline; a hormone (e.g., T-3 or T-4); a drug ofabuse (LSD, THC, barbituates, etc.); or a spore of Bacillus anthracis(anthrax). The analyte of interest can be in a liquid sample;alternatively, the analyte of interest can be in a dry (non-fluid)sample (e.g., a solid, such as a particulate sample, powder sample, orsoil sample).

In the methods of the invention, a fluid sample is assessed for thepresence or absence, or quantity, of an analyte of interest. The fluidcan be a fluid that wets the membrane material; that supports a reactionbetween the analyte of interest and the analyte binding agent, such asthe antibody/antigen reaction (i.e., does not interfere withantibody/antigen interaction); and that has a viscosity that issufficiently low to allow movement of the fluid by capillary action. Ina preferred embodiment, the fluid is an aqueous solution (such as abodily fluid). The fluid sample can be a fluid having relatively fewcomponents, for example, an aqueous solution containing the analyte ofinterest; alternatively, the fluid sample can be a fluid having manycomponents, such as a complex environmental sample (e.g., sewage, wastewater, groundwater, or other water sample), or a complex biologicalfluid (e.g., whole blood, plasma, serum, urine, cerebrospinal fluid,saliva, semen, vitreous fluid, synovial fluid, or other biologicalfluid). In a preferred embodiment in which the fluid is a biologicalfluid, the fluid is whole blood, plasma, or serum. If desired, the fluidsample can be diluted; for example, if a complex biological fluid isused as the fluid sample, it can be diluted with a solution (e.g., anaqueous solution).

If the analyte of interest is not in solution (e.g., the analyte ofinterest is in a dry or solid sample, as described above), it can beextracted, suspended, or dissolved into a fluid sample first. Forexample, if the analyte of interest is a nucleic acid, it can beextracted from cells of interest into a solution (e.g., an aqueoussolution, such as the buffer described below); in another example, ifthe analyte of interest is a powder or particulate material (e.g., apowder, a particulate, a soil sample, or spores), it can be suspended ordissolved into a solution (e.g., an aqueous solution, such as the bufferdescribed below) such as by obtaining a sample of the dry material(e.g., using a swab or other instrument) and placing the sample of drymaterial into the solution. Thus, a “fluid sample” can refer not only toa liquid sample to be assessed for an analyte of interest, but also to afluid sample in which a solid material (to be assessed for an analyte ofinterest) is extracted, suspended or dissolved.

The “analyte binding agent,” as used herein, refers to second member ofa binding pair as described above. The analyte binding agent is acompound that specifically binds to the analyte (the first member of thebinding pair), such as an antibody, a hapten or drug conjugate, areceptor, or another binding partner. In a preferred embodiment, theanalyte binding agent is an antibody to the analyte of interest.

Sandwich Assays

The “sandwich” assay of the invention utilizes a solid phase apparatus.The solid phase apparatus includes a membrane strip having anapplication point, a sample capture zone, and a control capture zone.The solid phase apparatus may optionally include a wicking pad followingthe control capture zone, and a sample pad adjacent to or covering theapplication point. The membrane strip can be made of a substance havingthe following characteristics: sufficient porosity to allow capillaryaction of fluid along its surface and through its interior; the abilityto allow movement of coated particles (e.g., analyte binding particles,as described below) or complexes of particles and analyte of interest(e.g., contacted analyte binding particles, as described below) bycapillary action (i.e., it must not block the particles or complexes ofparticles and analyte of interest); and the ability to be wet by thefluid containing the analyte (e.g., hydrophilicity for aqueous fluids,hydrophobicity for organic solvents). Hydrophobicity of a membrane canbe altered to render the membrane hydrophilic for use with aqueousfluid, by processes such as those described in U.S. Pat. No. 4,340,482,or U.S. Pat. No. 4,618,533, which describe transformation of ahydrophobic surface into a hydrophilic surface. Examples of membranesubstances include: cellulose, cellulose nitrate, cellulose acetate,glass fiber, nylon, polyelectrolyte ion exchange membrane, acryliccopolymer/nylon, and polyethersulfone. In a preferred embodiment, themembrane strip is made of cellulose nitrate (e.g., a cellulose nitratemembrane with a Mylar backing).

The “application point” is the position on the membrane where a fluidcan be applied. An “application pad” can also optionally be used; theapplication pad rests on the membrane, immediately adjacent to orcovering the application point. The application pad can be made of anabsorbent substance which can deliver a fluid sample, when applied tothe pad, to the application point on the membrane. Representativesubstances include cellulose, cellulose nitrate, cellulose acetate,nylon, polyelectrolyte ion exchange membrane, acrylic copolymer/nylon,polyethersulfone, or glass fibers. In one embodiment, the pad is aHemasep®-V pad (Pall Corporation). In another embodiment, the pad is aglass fiber pad. If a wicking pad is present, it can similarly be madefrom such absorbent substances.

The “sample capture zone” refers to a point on the membrane strip atwhich a “sample capture reagent” is immobilized (e.g., coated on and/orpermeated through the membrane). The sample capture reagent is ananalyte binding agent, such as those described above. The sample capturereagent need not be the same analyte binding agent as described above;however, the sample capture reagent also forms a binding pair with theanalyte of interest, in that it specifically and preferentially binds tothe analyte of interest. In a preferred embodiment, the sample capturereagent is an antibody directed against the analyte; it can be directedagainst the same epitope of the analyte as, or against a differentepitope of the analyte from, the epitope that binds to the antibodiesused as analyte binding agents coated on the particles.

The apparatus additionally includes a “control capture reagent”immobilized in a “control capture zone.” The control capture reagent isa reagent which reacts with the analyte binding particles, but whichdoes not interact with the analyte to be measured: for example, thecontrol capture reagent can react with the analyte binding agent on theanalyte binding agent-coated particles; with another material on theparticles; or with the particles themselves. For example, if the analytebinding agent is an antibody, the control capture reagent can be ananti-immunoglobulin antibody. In a preferred embodiment, the analytebinding agent is an antibody, and the control capture reagent is ananti-immunoglobulin antibody. The control capture reagent is immobilizedon the membrane (coated on and/or permeated in the membrane) in acontrol capture zone.

The control capture zone is positioned such that the sample capture zoneis between the application point and the control capture zone. In apreferred embodiment, the control capture zone is closely adjacent tothe sample capture zone, so that the dynamics of the capillary action ofthe components of the assay are similar (e.g., essentially the same) atboth the control capture zone and the sample capture zone. Although theyare closely adjacent, the control capture zone and the sample capturezone are also sufficiently spaced such that the particles arrested ineach zone can be quantitated individually (e.g., without cross-talk).Furthermore, in a preferred embodiment, the sample capture zone isseparated from the application point by a space that is a largedistance, relative to the small distance between the sample capture zoneand the control capture zone. The speed of the capillary front (theborder of the fluid moving through the membrane by capillary action) isinversely related to the distance of the capillary front from theapplication point of the fluid. Because particle capture is a ratelimiting step in the assay, the distance between the application point(where the capillary front mobilizes analyte binding particles) and thecapture zones (where particles are captured) must be sufficient toretard the speed of the capillary front to a rate that is slow enough toallow capture of particles when the capillary front reaches the samplecapture zone. In addition, the distance must be sufficiently large sothat the total time of migration (movement of the capillary frontthrough the entire membrane) is long enough to allow free analyte in afluid sample to bind to analyte binding particles. The optimal distancesbetween the components on the membrane strip can be determined andadjusted using routine experimentation.

The quantitative assay additionally uses a sample collection apparatus.A “sample collection apparatus,” as used herein, refers to an apparatusthat can be used for collection of the fluid sample or into which acollected fluid sample can be deposited or stored. The sample collectionapparatus can be any apparatus which can contain the analyte bindingparticles, as described below, and which to which can be added ameasured volume of fluid sample. Representative sample collectionapparatus include a sample tube, a test tube, a vial, a pipette orpipette tip, a syringe. In a preferred embodiment, the sample collectionapparatus is a pipette or pipette tip.

The sample collection apparatus contains a population of “analytebinding particles” which are coated with the analyte binding agent. Thepopulation of particles varies, depending on the size and composition ofthe particles, the composition of the membrane of the solid phaseapparatus, and the level of sensitivity of the assay. The populationtypically ranges approximately between 1×10³ and 1×10⁹, although feweror more can be used if desired. In a preferred embodiment, thepopulation is approximately 2×10⁸ particles.

The analyte binding particles are particles which can be coated with theanalyte binding agent (the second member of the binding pair). In apreferred embodiment, the analyte binding particles are liposomes,colloidal gold, organic polymer latex particles, inorganic fluorescentparticles or phosphorescent particles. In a particularly preferredembodiment, the particles are polystyrene latex beads, and mostparticularly, polystyrene latex beads that have been prepared in theabsence of surfactant, such as surfactant-free Superactive UniformAldehyde/Sulfate Latexes (Interfacial Dynamics Corp., Portland, Oreg.).

The size of the particles is related to porosity of the membrane (foranalytes in fluid samples) and also to the size of the analyte ofinterest (e.g., for particulate analytes): the particles must besufficiently small to be transported along the membrane by capillaryaction of fluid, and also (for solid, e.g., particulate analytes,sufficiently small for the complex of contacted analyte bindingparticles, as described below, to be transported along the membrane bycapillary action). The particles can be labeled to facilitate detection.The particles are labeled by a means which does not significantly affectthe physical properties of the particles; for example, the particles arelabeled internally (that is, the label is included within the particle,such as within the liposome or inside the polystyrene latex bead).Representative labels include luminescent labels; chemiluminescentlabels; phosphorescent labels; enzyme-linked labels; chemical labels,such as electroactive agents (e.g., ferrocyanide); and colorimetriclabels, such as dyes or fluorescent labels. In one embodiment, afluorescent label is used. In another embodiment, phosphorescentparticles are used, particularly “up-converting” phosphorescentparticles, such as those described in U.S. Pat. No. 5,043,265.

The particles are coated with an analyte binding agent that is a secondmember of the binding pair. As described above, the analyte bindingagent (second member of the binding pair) specifically andpreferentially binds to the analyte of interest (first member of thebinding pair). Representative analyte binding agents include antibodies(or fragments thereof); haptens; drug conjugates; receptors; or otherbinding partners. In one preferred embodiment, the analyte binding agentis an antibody to the analyte of interest. Antibodies can be monoclonalantibodies or polyclonal antibodies. The term “antibody”, as usedherein, also refers to antibody fragments which are sufficient to bindto the analyte of interest. Alternatively, in another embodiment,molecules which specifically bind to the analyte of interest, such asengineered proteins having analyte binding sites, can also be used(Holliger, P. and H. R. Hoogenbloom, Trends in Biotechnology 13:7-9(1995); Chamow, S. M. and A. Ashkenazi, Trends in Biotechnology14:52-60:1996)). In still another embodiment, if the analyte of interestis a drug, a hapten or other drug conjugate can be used as the analytebinding agent. Alternatively, in a further embodiment, a receptor whichbinds to the analyte can be used (e.g., if the analyte of interest is aligand). If the analyte is an antibody of known specificity, theparticles can be coated with the antigen against which theanalyte-antibody is directed, or can be coated with antibody to theanalyte-antibody. Furthermore, because the analyte and the analytebinding agent form a binding pair, compounds or molecules described asrepresentative analytes can also serve as analyte binding agents, andthose described as representative analyte binding agents can similarlyserve as analytes, as described herein.

The analyte binding particles contained within the sample collectionapparatus are stored in a stable form within the sample collectionapparatus. A “stable form,” as the term is used herein, indicates a formin which the particles do not significantly change in chemical makeup orphysical state during storage. The stable form can be a liquid, gel, orsolid form. In preferred embodiments, the analyte binding particlescontained within the sample collection apparatus are evaporativelydried; freeze-dried; and/or vacuum-dried.

In a particularly preferred embodiment, the sample collection apparatusis a pipette tip in which are vacuum-dried analyte binding particles.

To perform the assay, a fluid sample to be assessed for the presence ofthe analyte of interest, as described above, is used. In one embodiment,the fluid sample is introduced into (drawn into, poured into, orotherwise placed into) the sample collection apparatus. For example, inone embodiment, the fluid sample is drawn up into a sample collectionapparatus that comprises a pipette tip. Introduction of the fluid sampleinto the sample collection apparatus results in mixing of the fluidsample with the analyte binding particles, forming a “mixed fluidsample.” If the analyte binding particles are evaporatively-, freeze- orvacuum-dried, the introduction of the fluid sample into the samplecollection apparatus can result in rehydration and suspension of theanalyte binding particles in the fluid sample. A buffer (e.g, fordilution) is also introduced into the mixed fluid sample, forming a“buffered, mixed fluid sample.” The buffered, mixed fluid sample can beformed either by dispensing the mixed fluid sample into a “buffercontainer” (e.g., test tube) containing the buffer, or by introducingthe buffer into the sample collection apparatus prior to introducing thefluid sample. Alternatively, if the analyte of interest is a solid(e.g., a powder, a particulate; spore; or other particle, as describedabove), the fluid sample as described above can be prepared byintroducing the solid into the buffer container; in this embodiment, thebuffered, mixed fluid sample is formed by introducing the fluid sample(comprising the buffer) into the sample collection apparatus. In anotherembodiment, the buffer is introduced into the sample collectionapparatus, followed by introduction of the fluid sample into the samplecollection apparatus.

The buffer can be an aqueous fluid that supports a reaction between theanalyte of interest and the analyte binding agent (e.g., does notinterfere with antibody/antigen interaction); and that has a viscositythat is sufficiently low to allow movement of the fluid by capillaryaction. In one embodiment, the buffer contains one or more of thefollowing components: a buffering agent (e.g., phosphate); a salt (e.g.,NaCl); a protein stabilizer (e.g., BSA, casein, serum); and/or adetergent such as a nonionic detergent or a surfactant (e.g., one ormore of the following agents commonly available in surfactant tool kits:NINATE 411, Zonyl FSN 100, Aerosol OT 100%, GEROPON T-77, BIO-TERGEAS-40, STANDAPOL ES-1, Tetronic 1307, Surfnyol 465, Surfynol 485,Surfynol 104PG-50, IGEPAL CA210, TRITON X-45, TRITON X-100, TRITON X305,SILWET L7600, RHODASURF ON-870, Cremophor EL, TWEEN 20, TWEEN 80, BRIJ35, CHEMAL LA-9, Pluronic L64, SURFACTANT 10G, SPAN 60, CREL).Optionally, if desired, the buffer can contain a thickening agent. Suchcomponents for buffers are commercially available. Representativebuffers include, for example, saline, or 50 mM Tris-HCl, pH 7.2.Alternatively, water can be used in lieu of a buffered solution; as usedherein, the term “buffer” refers to either a buffered solution or towater.

To disperse the analyte binding particles further into the fluid sample,if desired, the sample collection apparatus into which the fluid sampleand the buffer has been introduced, or the buffer container into whichthe mixed fluid sample has been introduced, can be agitated (e.g.,vortexed, shaken, pipetted down and up, etc.).

In a preferred embodiment, the sample collection apparatus comprises apipette tip having vacuum-dried analyte binding particles within itstip; the fluid sample is drawn into the pipette, thereby rehydrating thedried analyte binding particles and forming a mixed fluid sample. In aparticularly preferred embodiment, the mixed fluid sample is introducedinto a buffer container, resulting in a buffered mixed fluid sample; thebuffered mixed fluid sample in the buffer container is pipetted up anddown using the sample collection apparatus, thereby further dispersingthe analyte binding particles.

If analyte of interest is present in the buffered, mixed fluid sample,binding occurs between the analyte and the analyte binding particles.“Binding” of analyte to the analyte binding particles indicates that theanalyte binding agent coated onto the particle is interacting with(e.g., binding to) analyte of interest. Analyte binding particles whichhave been maintained (incubated) under conditions allowing analyte inthe fluid (if present) to bind to the analyte binding particlesimmobilized in the application point are referred to herein as“contacted analyte binding particles”. Contacted analyte bindingparticles may or may not have analyte bound to the analyte bindingagent, depending on whether or not analyte is present in the fluidsample and whether analyte has bound to the analyte binding agent on theanalyte binding particles. Because there are multiple binding sites foranalyte on the analyte binding particles, the presence and theconcentration of analyte bound to analyte binding particles varies; theconcentration of analyte bound to the analyte binding particlesincreases proportionally with the amount of analyte present in the fluidsample, and the probability of an analyte binding particle beingarrested in the sample capture zone (as described below) similarlyincreases with increasing amount of analyte bound to the analyte bindingparticles. Thus, the population of contacted analyte binding particlesmay comprise particles having various amount of analyte bound to theanalyte binding agent, as well as particles having no analyte bound tothe analyte binding agent (just as the analyte binding particlesinitially have no analyte bound to the analyte binding agent).Furthermore, the degree of binding increases as the time factor of theconditions increases: while the majority of binding occurs within oneminute (e.g., 60 seconds, preferably less than 60 seconds (e.g., 45seconds, 30 seconds, or less), additional incubation (e.g., more thanone minute (2 minutes, 5 minutes, 10 minutes, 15 minutes) results inadditional binding.

The buffered, mixed fluid sample is applied to the application point ofthe membrane strip of the solid phase apparatus, or to the applicationpad, if present. After the membrane strip is contacted with thebuffered, mixed fluid sample, the membrane strip is maintained underconditions which allow fluid to move by capillary action to and throughthe membrane. The contacted analyte binding particles move through themembrane as a result of capillary action of the fluid from the buffered,mixed fluid sample, and the contacted analyte binding particles movealong the membrane to and through the “sample capture zone” on themembrane and subsequently to and through the “control capture zone.” Themembrane strip is maintained under conditions (e.g., sufficient time andfluid volume) which allow the contacted analyte binding particles tomove by capillary action along the membrane to and through the samplecapture zone and subsequently to the control capture zone, andsubsequently beyond the capture zones (e.g., into a wicking pad),thereby removing any non-bound particles from the capture zones.

The movement of some of the contacted analyte binding particles isarrested by binding of contacted analyte binding particles to the samplecapture reagent in the sample capture zone and subsequently by bindingof some of the contacted analyte binding particles to the controlcapture reagent in the control capture zone. In one preferredembodiment, the analyte binding agent is antibody to the antigen ofinterest, and the control capture reagent can be antibody againstimmunoglobulin of the species from which the analyte binding agent isderived. In this embodiment, the antibody to immunoglobulin should benon-cross reactive with other components of the sample: for example, ifa human sample is being tested, an antibody that does not react withhuman immunoglobulin can be used as the control capture reagent.

Sample capture reagent binds to contacted analyte binding particles bybinding to analyte which is bound to analyte binding agent on thecontacted analyte binding particles. The term, “sample-reagent-particlecomplexes”, as used herein, refers to a complex of the sample capturereagent and contacted analyte binding particles. Contacted analytebinding particles are arrested in the sample capture zone, forming thesample-reagent-particle complexes, due to capture of contacted analytebinding particles by interaction of analyte with sample capture reagentin the sample capture zone.

Control capture reagent binds to contacted analyte binding particles bybinding to analyte binding agent on the contacted analyte bindingparticles. The term, “control-reagent-particle complexes,” as usedherein, refers to a complex of the control capture reagent and contactedanalyte binding particles. Contacted analyte binding particles arearrested in the control capture zone, forming thecontrol-reagent-particle complexes, due to capture of contacted analytebinding particles by interaction of analyte binding particles withcontrol capture reagent in the control capture zone. As indicated above,the control capture reagent interacts with the analyte binding particles(e.g., with the analyte binding agent on the analyte bindingagent-coated particles, or another material on the particles, or withthe particles themselves), but not with the analyte itself.

Capillary action subsequently moves any contacted analyte bindingparticles that have not been arrested in either the sample capture zoneor the control capture zone, onwards beyond these zones, therebyremoving any particles that have not been arrested.

In a preferred embodiment, the fluid moves any contacted analyte bindingparticles that have not been arrested, into a wicking pad which followsthe control capture zone.

If desired, a secondary wash step can be used. A buffer (e.g., thebuffer described above) can be applied at the application point afterthe buffered, mixed fluid sample has soaked in to the membrane or intothe application pad, if present. The secondary wash step can be used atany time thereafter, provided that it does not dilute the buffered,mixed fluid sample. A secondary wash step can contribute to reduction ofbackground signal when the analyte binding particles are detected, asdescribed below.

The amount of analyte binding particles arrested in the sample capturezone (sample-reagent-particle complexes) is then detected using anappropriate means for the type of label used on the analyte bindingparticles. In a preferred embodiment, the amount is detected by anoptical method, such as by measuring the amount of fluorescence of thelabel of the analyte binding particles. Alternatively, the amount ofsample-reagent-particle complexes can be detected using electricalconductivity or dielectric (capacitance). Alternatively, electrochemicaldetection of released electroactive agents, such as indium, bismuth,gallium or tellurium ions, as described by Hayes et al. (AnalyticalChem. 66:1860-1865 (1994)) or ferrocyanide as suggested by Roberts andDurst (Analytical Chem. 67:482-491 (1995)) can be used. For example, ifliposomes are used, ferrocyanide encapsulated within the liposome can bereleased by addition of a drop of detergent at the capture zone, and thereleased ferrocyanide detected electrochemically (Roberts and Durst,id). If chelating agent-protein conjugates are used to chelate metalions, addition of a drop of acid at the capture zone will release theions and allow quantitation by anodic stripping voltametry (Hayes etal., id.). Similarly, the amount of analyte binding particles arrestedin the control capture zone is detected in the same manner as the amountof analyte binding particles in the sample capture zone.

In one embodiment, the detected amount of analyte binding particles isrepresented by a curve that is directly related to the amount of labelpresent at positions along the solid phase (e.g., the membrane strip).For example, the detected amounts of particles at each position on themembrane strip (e.g., at the sample capture zone and the control capturezone, and/or areas in between or adjacent to the sample capture zone andthe control capture zone, and/or other areas of the membrane strip) canbe determined and plotted as a function of the distance of the positionalong the membrane strip. The amount of particles can then be calculatedas a function of the area under the curve, which is related to theamount of label present.

A corrected analyte binding particle amount is then determined, and theamount of analyte can then be determined from the corrected analytebinding particle amount using appropriate calculation. The correctedanalyte binding particle amount is based on the amount of analytebinding particles arrested in the sample capture zone and in the controlcapture zone. For example, in one embodiment, the corrected analytebinding particle amount is determined as a ratio (R) of the analytebinding particle amount present in the sample capture zone to theanalyte binding particle amount present in the control capture zone. Theamount of analyte present can be then determined from the correctedanalyte binding particle amount (the ratio), utilizing a standard curve.The standard curve is generated by preparing a series of controlsamples, containing known concentrations of the analyte of interest inthe fluid in which the analyte is to be detected (for example, such asserum depleted of the analyte). The assay is then performed on theseries of control samples; the value of R is measured for each controlsample; and the R values are plotted as a function of the concentrationof analyte included in the control sample. Samples containing an unknownamount of analyte (the “test samples”) are assayed by measuring thevalue of R for the test sample, and the concentration of analyte in thetest sample is determined by referring to the standard curve. As above,one standard curve can be generated and used for all test samples in alot (e.g., for all test samples using a specified preparation of testreagents); it is not necessary that the standard curve be re-generatedfor each test sample. In another embodiment, the corrected analytebinding particle amount is determined as a ratio (R) of the amount ofthe analyte binding particle amount present in the sample capture zone,to the sum of the analyte binding particle amount present in the controlcapture zone and the analyte binding particle amount present in thesample capture zone. The amount of analyte present can be thendetermined from corrected analyte binding particle amount (the ratio),utilizing a standard curve. Alternatively, other ratios and/or standardcurves can also be used to determine the amount of analyte in thesample. In addition, if desired, the amount of label that is present inthe background can be subtracted from the analyte binding particleamount present in the sample capture zone and the analyte bindingparticle amount present in the control capture zone prior to calculationof the ratio (R).

“Competitive” or “Inhibition” Assays

The “competitive” or “inhibition” assay of the invention, like the“sandwich” assays, utilizes a solid phase apparatus including a membranestrip, as described above, that includes an application point, a samplecapture zone, and a control capture zone. The membrane strip mayoptionally include a wicking pad following the control capture zone, anda sample pad preceding the application point. As before, the“application point” is the position on the membrane where a fluid sampleis applied. This embodiment also utilizes a sample collection apparatus,as described above. The sample collection apparatus for the competitive(inhibition) assay contains a population of “analyte coated particles”which are coated with the analyte of interest (in lieu of being coatedwith an analyte binding agent, as described for the “sandwich” assays)or with an analog of the analyte of interest. An “analog” of theanalyte, as used herein, is a compound that has similar bindingcharacteristics as the analyte, in that is forms a binding pair with theanalyte-binding agent as described above. The analyte or analog of theanalyte can be coated directly on the particles, or can be indirectlybound to the particles. As used below, the term “analyte coatedparticles” can refer to particles that are coated either with analyte ofinterest or with an analog of the analyte of interest. As above withregard to the sandwich assay, the population of particles varies,depending on the size and composition of the particles, the compositionof the membrane of the solid phase apparatus, and the level ofsensitivity of the assay.

As above, the sample capture zone refers to a point on the membranestrip at which a sample capture reagent is immobilized. The samplecapture reagent is an analyte binding agent, such as those describedabove. The sample capture reagent need not be the same analyte bindingagent as described above; however, the sample capture reagent also formsa binding pair with the analyte of interest, in that it specifically andpreferentially binds to the analyte of interest. As above, in apreferred embodiment, the sample capture reagent is an antibody directedagainst the analyte; it can be directed against the same epitope of theanalyte as, or against a different epitope of the analyte from, theepitope that binds to the antibodies used as analyte binding agentscoated on the particles.

The apparatus additionally includes a control capture reagent, asdescribed above, that reacts with the analyte coated particles, but doesnot interact with the analyte to be measured: for example, the controlcapture reagent can react with another material on the particles (e.g.,a carrier for the analyte that is bound to the particles; an antibody);or with the particles themselves. In a preferred embodiment, the samplecapture reagent and the control capture agent are both antibodies. Thecontrol capture reagent is immobilized on the membrane (coated on and/orpermeated in the membrane) in the control capture zone.

The components of the competitive assay are positioned in a similarmanner as described above with regard to the “sandwich” assay. Forexample, in a preferred embodiment, the control capture zone is closelyadjacent to the sample capture zone, so that the dynamics of thecapillary action of the components of the assay are similar (e.g.,essentially the same) at both the control capture zone and the samplecapture zone; and yet the control capture zone and the sample capturezone are also sufficiently spaced such that the particles arrested ineach zone can be quantitated individually. Furthermore, in a preferredembodiment, the sample capture zone is separated from the applicationpoint by a space that is a large distance, relative to the smalldistance between the sample capture zone and the control capture zone,in order to ensure that the speed of the capillary front is sufficientlyslow to allow capture of particles, and the total time of migration issufficiently long to allow for binding of analyte to the sample capturereagent.

To perform the competitive assay, a fluid sample to be assessed for thepresence of the analyte of interest, as described above, is used. In oneembodiment, the fluid sample is introduced into (drawn into, pouredinto, or otherwise placed into) the sample collection apparatus. Forexample, in one embodiment, the fluid sample is drawn up into a samplecollection apparatus that comprises a pipette tip. Introduction of thefluid sample into the sample collection apparatus results in mixing ofthe fluid sample with the analyte coated particles, forming a “mixedfluid sample.” If the analyte coated particles are evaporatively-,freeze- or vacuum-dried, the introduction of the fluid sample into thesample collection apparatus can result in rehydration and suspension ofthe analyte binding particles in the fluid sample. A buffer (e.g., asdescribed above) is also introduced into the mixed fluid sample, forminga “buffered, mixed fluid sample.” The buffered, mixed fluid sample canbe formed either by dispensing the mixed fluid sample into a “buffercontainer” (e.g., test tube) containing the buffer, or by introducingthe buffer into the sample collection apparatus prior to introducing thefluid sample. In another embodiment, the buffer is introduced into thesample collection apparatus, followed by introduction of the fluidsample into the sample collection apparatus. Alternatively, if theanalyte of interest is a solid (e.g., a powder, a particulate; spore; orother particle, as described above), the fluid sample as described abovecan be prepared by introducing the solid into the buffer container; inthis embodiment, the buffered, mixed fluid sample is formed byintroducing the fluid sample (comprising the buffer) into the samplecollection apparatus.

To disperse the analyte coated particles further into the fluid sample,if desired, the sample collection apparatus into which the fluid sampleand the buffer has been introduced, or the buffer container into whichthe mixed fluid sample has been introduced, can be agitated (e.g.,vortexed, shaken, pipetted down and up, etc.).

In a preferred embodiment, the sample collection apparatus comprises apipette tip having vacuum-dried analyte coated particles within its tip;the fluid sample is drawn into the pipette, thereby rehydrating thedried analyte coated particles and forming a mixed fluid sample. In aparticularly preferred embodiment, the mixed fluid sample is introducedinto a buffer container, resulting in a buffered mixed fluid sample; thebuffered mixed fluid sample in the buffer container is pipetted up anddown using the sample collection apparatus, thereby further dispersingthe analyte coated particles.

The buffered, mixed fluid sample is applied to the application point ofthe membrane strip of the solid phase apparatus, or to the applicationpad, if present. After the membrane strip is contacted with thebuffered, mixed fluid sample, the membrane strip is maintained underconditions which allow fluid to move by capillary action to and throughthe membrane. The analyte coated particles (and analyte, if present inthe sample) move through the membrane as a result of capillary action ofthe fluid from the buffered, mixed fluid sample, to and through the“sample capture zone” on the membrane and subsequently to and throughthe “control capture zone.” The membrane strip is maintained underconditions (e.g., sufficient time and fluid volume) which allow theanalyte coated particles to move by capillary action along the membraneto and through the sample capture zone and subsequently to the controlcapture zone, and subsequently beyond the capture zones (e.g., into awicking pad), thereby removing any non-bound particles from the capturezones.

The movement of some of the analyte coated particles is arrested bybinding of analyte coated particles to the sample capture reagent in thesample capture zone, and subsequently by binding of some of the analytecoated particles to the control capture reagent in the control capturezone. The analyte coated particles compete with analyte (if present) inthe sample for binding to the sample capture reagent. The sample capturereagent binds to analyte coated particles by binding to analyte on theanalyte coated particles. The term,“sample-reagent-analyte-coated-particle complexes”, as used herein,refers to a complex of the sample capture reagent and analyte coatedparticles. The analyte coated particles are arrested in the samplecapture zone, forming the sample-reagent-analyte-coated-particlecomplexes, due to capture of the analyte coated particles by interactionof the analyte on the particles with the sample capture reagent in thesample capture zone.

The control capture reagent binds to analyte coated particles by bindingto any component of the analyte-coated particles except the analyteitself. The term, “control-reagent-analyte-coated particle complexes,”as used above, refers to a complex of the control capture reagent andanalyte coated particles. As above, the analyte coated particles arearrested in the control capture zone, forming thecontrol-reagent-analyte-coated particle complexes, due to capture of theanalyte coated particles by interaction of the analyte binding particleswith the control capture reagent in the control capture zone.

Capillary action subsequently moves any analyte coated particles thathave not been arrested in either the sample capture zone or the controlcapture zone, onwards beyond the control capture zone. In a preferredembodiment, the fluid moves any contacted analyte coated particles thathave not been arrested in either capture zone into a wicking pad whichfollows the control capture zone.

The amount of analyte coated particles arrested in the sample capturezone is then detected. The analyte coated particles are detected usingan appropriate means for the type of label used on the analyte coatedparticles. In a preferred embodiment, the amount of analyte coatedparticles is detected by an optical method, such as by measuring theamount of fluorescence of the label of the analyte-binding particles.The amount of analyte coated particles arrested in the control capturezone is detected in the same manner as the amount of analyte coatedparticles in the sample capture zone. In one embodiment, as describedabove, the amount of analyte coated particles is represented by a curvethat is directly related to the amount of label present at positionsalong the solid phase (e.g., the membrane strip). For example, theamount of particles at each position on the membrane strip (e.g., at thesample capture zone and the control capture zone, and/or areas inbetween or adjacent to the sample capture zone and the control capturezone, and/or other areas of the membrane strip) can be determined andplotted as a function of the distance of the position along the membranestrip. The amount of particles can then be calculated as a function ofthe area under the curve, which is related to the amount of labelpresent.

A corrected analyte coated particle amount is determined, and the amountof analyte can then be determined from the corrected analyte coatedparticle amount using appropriate calculation. The corrected analytecoated particle amount is based on the amount of analyte coatedparticles arrested in the sample capture zone and in the control capturezone. For example, in one embodiment, the corrected analyte coatedparticle amount is inversely proportional to a ratio (R) of theanalyte-coated particle amount present in the sample capture zone to theanalyte-coated particle amount present in the control capture zone. Theamount of analyte present can be then determined from the correctedanalyte coated particle amount (the ratio), utilizing a standard curve.The standard curve is generated by preparing a series of controlsamples, containing known concentrations of the analyte of interest inthe fluid in which the analyte is to be detected (such as serum depletedof the analyte). The assay cam then performed on the series of controlsamples; the value of R is measured for each control sample; and the Rvalues are plotted as a function of the concentration of analyteincluded in the control sample. Samples containing an unknown amount ofanalyte (the “test samples”) are assayed by measuring the value of R forthe test sample, and the concentration of analyte in the test sample isdetermined by referring to the standard curve. As above, one standardcurve can be generated and used for all test samples in a lot (e.g., forall test samples using a specified preparation of test reagents); it isnot necessary that the standard curve be re-generated for each testsample. In another embodiment, the corrected analyte coated particleamount is inversely proportional to a ratio (R) of the amount of theanalyte coated particle amount present in the sample capture zone, tothe sum of the analyte coated particle amount present in the controlcapture zone and the analyte coated particle amount present in thesample capture zone. The amount of analyte present can be thendetermined from corrected analyte coated particle amount (the ratio),utilizing a standard curve. Alternatively, other ratios and/or standardcurves can also be used to determine the amount of analyte in thesample. In addition, if desired, the amount of label that is present inthe background can be subtracted from the analyte coated particle amountpresent in the sample capture zone and the analyte coated particleamount present in the control capture zone prior to calculation of theratio (R).

Benefits of the Invention

The methods of the invention provide assays with enhanced sensitivity,when compared with assays in which the analyte binding particles areimbedded within the membrane of the solid phase apparatus. For thesandwich assays, for example, because the fluid sample to be assayed forthe analyte of interest is mixed with the analyte binding particlesprior to application to the membrane, there is a longer time for theanalyte of interest to bind to the analyte binding particles prior tothe capture reaction which occurs in the membrane. Furthermore, becausethe interaction between the analyte of interest and the analyte bindingparticles occurs in the fluid phase, it allows more efficient bindingbecause of greater mobility of the particles, than the same interactionbetween analyte of interest and analyte binding particles would be inthe matrix of the membrane of the solid phase apparatus. Also, withregard to both the sandwich and the competitive assays, a greater numberof particles can be included in a fluid collection apparatus than wouldbe possible to embed in a solid phase apparatus; the greater numberfurther enhances the sensitivity of the reaction. In addition, becausethe analyte binding particles (or analyte coated particles) aredispersed in the buffered, mixed fluid sample prior to application ofthe buffered, mixed fluid sample to the solid phase membrane, theparticles pass over the capture zone(s) in a continuous manner throughthe capillary action of the fluid, rather than in a quick wave on thecrest of a fluid front. As a result, a lower concentration of particlesflows through the capture zone(s) for a longer time: thus the timeduring which particles can be “captured” is effectively increased, whilethe amount of particles that pass through the capture zone(s) iseffectively lowered, thereby avoiding the blocking of capture of someparticles by others which occurs when the particles pass on the crest ofa fluid front.

Although the assays of the invention have been described particularly inrelation to immunoassays, the assays can similarly be used with otherbinding pairs as described above (e.g., nucleic acids, receptor-ligands,lectin-sugars), using the same methods as described above with thedesired components as the analyte and the and the analyte binding agent.

Kits of the Invention

The invention also includes kits for use in the methods describedherein. Kit components can include: first and/or second members of aspecific binding pair, buffers and/or buffer containers, fluidcollection means, one or more solid phase apparatus (optionallycomprising an application pad and/or wicking pad), at least one samplecollection apparatus, one or more buffer containers, control samples forgeneration of a standard curve and/or other standard curve information,analyte binding particles, analyte coated particles, and/or controlparticles, capture reagents, antibodies, tools to assist in collectingof samples to be assessed for analyte of interest (e.g., swabs),disposal apparatus (e.g., biohazard waste bags), and/or otherinformation or instructions regarding the sample collection apparatus(e.g., lot information, expiration date, etc.). For example, in oneembodiment, a kit comprises at least one sample collection apparatushaving analyte binding particles within it; in a preferred embodiment, akit comprises at least one pipette tip having evaporatively-dried,vacuum-dried or freeze-dried analyte binding particles therein. Inanother embodiment, a kit comprises at least one solid phase apparatusas described herein and at least one sample collection apparatus. Inanother preferred embodiment, a kit comprises at least one pipette; atleast one or more pipette tips having evaporatively-dried, vacuum-driedor freeze-dried analyte binding particles therein; and at least onesolid phase apparatus. This preferred embodiment can also optionallycontain information regarding the standard curve, lot information,and/or expiration date relating to the analyte binding particles in thepipette tips. In yet another preferred embodiment, a kit comprises atleast one sample collection apparatus; at least one pipette tip havingdried analyte binding particles thereon; at least one solid phaseapparatus; and at least one buffer container. This preferred embodimentcan also optionally contain buffer within the buffer container; and tool(e.g., a swab) for collection of a solid sample.

The invention is further illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLE 1 TITRATION OF LATEX INTO BUFFER, COMPARED WITH CONTROL STRIPS

Materials and Methods

Experiments were set up as follows. To prepare the solid phase apparatus(“test cartridge”), nitrocellulose membrane pre-attached to Mylarbacking was used. A specific antibody (sample capture reagent) wasapplied to the membrane at the sample capture zone, and an internalcontrol antibody (control capture reagent) was applied to the membraneat the control capture zone. After the antibodies were applied, theprotein binding sites on the membrane were blocked by treatment withPoly Vinyl Alcohol (PVA), and then the membranes were washed and dried.The membrane was then cut into 5 mm wide strips, perpendicularly to thelines of antibody applied. The strips were then assembled into testcartridges (solid phase apparatus) along with two pads: a) anapplication pad at the proximal end that functions as a filter toseparate blood cells (if whole blood is used), or alternatively, a glassfiber pad (to act as a sample holding pad for the anthrax test); and b)a wicking pad at the distal end (beyond the antibody lines) of glassfiber to act as an absorbent pad to soak up fluid that travels throughthe membrane from the proximal end where the sample is added to thedistal end.

The test cartridge was press fit together with an opening in thecartridge and a “well” above the sample pad, pressing into the pad, sothat a liquid sample was retained until it could be absorbed into thepad and membrane. The bottom of the cartridge had a “window” where themembrane lower surface (the Mylar backing) is exposed so that opticalreadings can be taken after the test is run. There were no otheropenings in the cartridge, although it was not hermetically sealed. Thecartridge was desiccated and then sealed into a foil pouch with a smalldesiccant pouch.

The pipette tip (sample collection apparatus) was configured as follows:commercially available pipette tips for use with automatic pipettes ofvarious types were placed into a rack that holds them in a verticalorientation a 12×8 matrix with 9 mm centers. An automated systemdelivered 5 microliters of an antibody-coated, dyed latex suspensioninto each tip. The suspension was delivered as a small (2 mm) dot about1 cm from the small end of the tip. The suspension was in a solution oftrehalose (sugar), in order to make the suspension viscous so that itstays in place when delivered to the tip; to help stabilize the proteinwhen it is dried; and to act as a “glue” so that the dried spot stays inplace. The tips were then dried under vacuum conditions, and once dried,were placed into the same pouch as the cartridge (one of each) prior tosealing.

Buffer vials were configured as follows: the buffer vial had a tightfitting cap and contained a pre-measured amount of diluent. Once thediluent vial was filled, it was capped and put into the test kit. Theamount and composition of the buffer varied for each different type ofassay. The quantity was adjusted so that the sample is diluted to alevel that is appropriate for each test. For instance, a myoglobin testuses a 1:10 dilution of blood, the troponin I test uses a 1:3 dilutionof blood. The anthrax test uses sufficient buffer to suspend the sampleand latex after losses in the test are accounted for. The buffer can bewater based and include several different salts, buffers and detergents.For example, representative buffers for representative analytes includethe following: for myoglobin, a buffer including 122 mM PB, 100 mM NaCl,3.3% BSA, 1.1% Cremophor EL, 0.05% ProClin300, pH 7.2 can be used; forCKMB, a buffer including 115 mM PB, 115 mM NaCl, 2.3% BSA, 0.25%Surfynol 104PG-50, 0.3% Casein, 40 mM Phe, 0.5% Sheep serum, 0.05%ProClin 300, pH 7.2 can be used; for TnI, a buffer including 124 mM PB,124 mM NaCl, 3.24% BSA, 0.76% Surfactant 10G, 0.54% casein, 405 mM GHCl,40 mM Phe, 10% Goat serum, pH 7.2 can be used; for anthrax, a bufferincluding 138 mM PB, 138 mM NaCl, 3.6% BSA, 0.84% Surfactant 10G, 0.6%casein, 0.05% High viscosity Methyl cellulose, 0.05% ProClin 300, pH 7.2can be used (abbreviations: PB=Phosphate Buffer, NaCl=Sodium Chloride,BSA=Bovine Serum Albumin, Phe=Phenylalanine).

To perform the assay for a blood sample, the following procedure wasfollowed:

A sample of blood was pipetted into the buffer vial, either with anytype of pipette OR with the pipette tip with the latex dot (samplecollection apparatus). This was a simple liquid transfer operation. Thepipette tip with the latex dot was then used to mix the sample andbuffer, by repeatable pipetting the liquid up and down at least 10times. During this mixing operation, the liquid came into contact withthe latex dot and caused it to rehydrate and become suspended in thediluent-sample matrix. Once suspended, the antibody on the latex beganto bind to antigen (if present) in the sample. The pipette (with thesame tip as above) was then used to transfer a portion of the bufferedmixture to the sample well in the cartridge. The cartridge was thenplaced into the reader where the following occurs: a) the diluent beganto soak into the sample pad where blood calls are retained. The liquidflowed through the pad and contacted the nitrocellulose membrane. Theliquid (with suspended latex) flowed by capillary action along thelength of the membrane to the absorbent pad which becomes wetted. Whenthe latex reached the test line (sample capture zone), if there is anyantigen in the sample some would be captured by the immobilized antibody(sample capture reagent). If the antigen had also been bound by theantibody on the latex, the latex would also be captured, by virtue ofthe antigen being sandwiched between the two antibodies. Latex that wasnot captured at the test line continued to flow and next encountered theinternal control antibody line (control capture zone). This antibody wasdirected towards the material (antibody or other material) on the latexitself. A portion of the latex was captured at the internal control linedue to this capture reaction. The remaining liquid and unbound latexcontinued to the distal end of the strip and was retained in the wickingpad. The liquid flow stopped when the pad is saturated OR when thesample was fully absorbed into the sample pad and capillary pressure wasequilibrated. The reader then determined the amount of latex at the testline and in the internal control line and applied a ratio method todetermine the quantity of antigen in the sample by comparison to astandard curve determined for that lot of test cartridges.

To run the assays for samples with particulate antigens (i.e. anthraxspores), samples are taken as follows. If the material is to be samplesfrom a surface (e.g., a table top) the following procedure was used: aswab was introduced into the buffer vial to dampen the swab tip; theswab was then used to gently scrub the surface to be tested; the swabwas then put back into the buffer vial and twirled to allow release ofthe particulate material into the diluent; the swab was removed anddiscarded. The test procedure above was then followed. If the materialto be tested was in liquid form (e.g., a liquid suspension of particles)the following was performed: a sample (10 to 30 microliters) of the testliquid was applied to the dry swab OR the dry swab was immersed into thetest liquid; the Swab was then immersed in the diluent liquid and gentlytwirled to release the particles; the swab was removed and discarded.The test procedure was followed as above. If the material to be testedwas in powder or granular form the following was performed: a smallsample of the powder was added to the diluent by either scooping a smallamount of the material into the vial OR by touching a wet or dry swabtip to the powder and then placing it into the vial of diluent. The testprocedure was followed as above.

Results

Using the methods described above, assays were run to compare resultsobtained by titrating latex particles into buffer (diluent) using driedlatex in pipette tips, with results obtained by coating latex particlesonto the membrane strips (at a latex application site “LAS”) andallowing the fluid of the sample to move by capillary action through theLAS prior to movement through the sample capture zone and the controlcapture zone. The samples were horse serum standards and blood M5 atbaseline (O) and 0.5 ng/ml TnI. The buffer (diluent) was TnI diluentwith 200 mM GHCl+10% goat serum, with 0, 0.5×, 1×, 2×, 4×, or 8× latexparticles. Results are shown in Table 1. TABLE 1 Effect of TitratingLatex Particles in Pipette with Buffer, Compared with Latex Particles atLAS on Membrane Strip S:B S-B Diluent/Strips Conc R10 SD CV R10 R10Horse Serum Standards R10 Values Control 0 0.041 0.006 15.1% 1.32 0.003with LAS strips 0.5 0.054 0.004 6.6% 0.5x Latex 0 0.036 0.004 11.9% 1.980.030 with no LAS strips 0.5 0.071 0.001 1.8% 1x Latex 0 0.040 0.0011.3% 1.90 0.031 with no LAS strips 0.5 0.076 0.004 5.9% 2x Latex 0 0.0490.002 3.6% 1.83 0.032 with no LAS strips 0.5 0.090 0.007 8.1% 4x Latex 00.065 0.001 2.3% 1.71 0.035 with no LAS strips 0.5 0.110 0.009 8.1% 8xLatex 0 0.101 0.003 2.6% 1.39 0.034 with no LAS strips 0.5 0.141 0.0021.7% Blood M5 R10 Values Control 0 0.040 0.007 16.8% 1.35 0.003 with LASstrips 0.5 0.054 0.004 7.4% 0.5x Latex 0 0.029 0.001 5.0% 1.85 0.023with no LAS strips 0.5 0.054 0.001 1.3% 1x Latex 0 0.033 0.001 4.4% 2.190.033 with no LAS strips 0.5 0.073 0.005 6.4% 2x Latex 0 0.049 0.00613.3% 1.78 0.028 with no LAS strips 0.5 0.087 0.004 4.5% 4x Latex 00.082 0.010 12.7% 1.66 0.032 with no LAS strips 0.5 0.135 0.011 8.5% 8xLatex 0 0.158 0.006 3.8% 1.33 0.030 with no LAS strips 0.5 0.210 0.0167.4%

It was observed that using the latex diluents at 0.5×, 1×, 2×, and 4×resulted in a significant increase in signal:background (S:B) comparedto the control LAS strips. The increase in S:B was due to an increase inspecific binding of the positive sample and similar background of thezero sample. This resulted in an increase in sensitivity of the system.As the amount of latex increased, the raw TL and ISL signals increasedaccordingly. The 1× latex diluent gave comparable TL and ISL signals tothe control LAS strips. Thus, it was concluded that the latex diluentgave higher sensitivity than the control LAS strips in RAMP assays.

EXAMPLE 2 Comparison of 1× Latex Diluent vs. LAS Strips in StandardCurve

Using the methods described above in Example 1, assays were run tocompare results obtained by titrating latex particles into buffer(diluent) using dried latex in pipette tips, with results obtained bycoating latex particles onto the membrane strips (at a latex applicationsite “LAS”) as described above. The samples were horse serum standardsand blood M35 spiked at 0, 0.1, 0.2, 0.5, 1 and 3 ng/ml. The buffer(diluent) was TnI diluent with 180 mM GHCl+10% goat serum, with orwithout 1× latex particles. Results are shown in Table 2. TABLE 2 FullStandard Curves in Horse Serum and Blood Using 1X Latex Particle Diluentand Full Length Membrane Strips R10 R10 Sample Tnl Conc R10 SD CV S:BS-B R10 Values - 1X Latex Diluent with No LAS Strips (Full-length) HSStds 0.0 0.018 0.001 5.6% — — 0.1 0.026 0.002 8.3% 1.45 0.005 0.2 0.0300.001 2.7% 1.66 0.010 0.5 0.049 0.001 1.7% 2.72 0.029 1.0 0.071 0.0033.8% 3.92 0.049 3.0 0.164 0.013 8.0% 9.03 0.131 Blood M35 0.0 0.0170.002 10.1% — — 0.1 0.023 0.002 7.7% 1.37 0.003 0.2 0.026 0.001 5.3%1.55 0.006 0.5 0.043 0.003 5.9% 2.50 0.021 1.0 0.064 0.003 4.2% 3.740.042 3.0 0.145 0.008 5.4% 8.55 0.119 Avg CV 5.7% R10 Values - ControlDiluent (no Latex) with Control strips (with LAS) HS Stds 0.0 0.0340.001 2.5% — — 0.1 0.044 0.002 4.7% 1.31 0.007 0.2 0.042 0.004 10.0%1.24 0.003 0.5 0.054 0.005 8.3% 1.62 0.015 1.0 0.063 0.008 12.1% 1.890.021 3.0 0.106 0.010 9.5% 3.14 0.061 Blood M35 0.0 0.029 0.002 7.8% — —0.1 0.035 0.005 13.6% 1.19 −0.001 0.2 0.033 0.003 9.8% 1.13 −0.002 0.50.042 0.002 5.1% 1.46 0.009 1.0 0.055 0.003 5.1% 1.89 0.021 3.0 0.1010.006 5.7% 3.50 0.064 Avg CV 8.3%

It was observed that the 1× latex diluent resulted in a consistentincrease in S:B over an entire standard curve range in both diluent andblood samples compared to the control LAS strips. The improvedsensitivity was observed not only in the higher S:B but also in thediscrimination of the 0.1 ng/ml point from zero in blood using the latexdiluent whereas with the LAS strips, there was not clear discriminationfrom zero until 0.5 ng/ml. These results confirmed the significantimprovement in sensitivity of the RAMP system using latex diluentcompared to LAS strips.

EXAMPLE 3 Titration of Latex Diluent vs. Control LAS Strips

Using the methods described above in Example 1, assays were run tocompare results obtained by titrating latex particles into buffer(diluent) using dried latex in pipette tips, with results obtained bycoating latex particles onto the membrane strips (at a latex applicationsite “LAS”) as described above. The samples were 0 and 2000 ng/mlanthrax spores (Bacillus anthracis). The buffer (diluent) was TnIdiluent with or without 1×, 3× or 6× latex particles. Results are shownin Table 3. TABLE 3 Assay for Anthrax Using Latex Particle Diluent andFull Length Membrane Strips BA Samples R10 Values S:B S-B Diluent/StripsConc R10 SD CV R10 R10 Control Diluent (No latex) 0 0.010 0.001 11.3%1.54 0.002 with LAS strips 2000 0.016 0.003 17.2% 1x Latex Diluent 00.026 0.008 30.8% 2.46 0.030 with no LAS strips 2000 0.064 0.000 0.0% 3xLatex Diluent 0 0.018 0.002 11.1% 3.28 0.036 with no LAS strips 20000.059 0.003 5.1% 6x Latex Diluent 0 0.020 0.001 5.0% 3.30 0.040 with noLAS strips 2000 0.066 0.005 7.6%

It was observed that using the latex diluent at 1×, 3×, and 6× resultsin a significant increase in S:B compared to the control LAS strips forthe anthrax assay. The S:B were higher for the 3× and 6× latex diluentsthan the 1× latex diluent. These results confirmed that the improvementin sensitivity of the RAMP system using latex diluent compared to LASstrips applied to the anthrax assay.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-22. (canceled)
 23. A method for quantitatively measuring the amount ofan analyte of interest in a fluid sample, comprising: a) providing asolid phase apparatus comprising a membrane strip comprising anapplication point, a sample capture zone and a control capture zone,wherein the sample capture zone is between the application point and thecontrol capture zone; b) providing a pipette containing a population offreeze dried analyte binding particles, wherein the analyte bindingparticles are coated with an analyte binding agent; c) either i)introducing the fluid sample into the pipette, producing a mixed fluidsample, and subsequently introducing a buffer into the mixed fluidsample; ii) introducing a buffer into the pipette and subsequentlyintroducing the fluid sample; or iii) forming the fluid sample byintroducing a solid into a buffer, and subsequently introducing thefluid sample into the sample collection apparatus, thereby producing abuffered, mixed fluid sample comprising contacted analyte bindingparticles; d) applying the buffered, mixed fluid sample to theapplication point of the membrane strip; e) maintaining the membranestrip under conditions which allow fluid to transport contacted analytebinding particles by capillary action through the strip to and throughthe sample capture zone, the sample capture zone having a sample capturereagent immobilized thereon; and allow contacted analyte bindingparticles to bind to the sample capture reagent; f) further maintainingthe membrane strip under conditions which allow the fluid in the sampleto transport contacted analyte binding particles by capillary actionthrough the strip to and through the control capture zone, the controlcapture zone having a control capture reagent immobilized thereon; andallow contacted analyte binding particles to bind to the control capturereagent; g) further maintaining the membrane strip under conditionswhich allow the fluid in the sample to transport any contacted analytebinding particles not bound to the sample capture reagent or to thecontrol capture reagent by capillary action beyond the control capturezone; h) determining the amount of contacted analyte binding particlesin the sample capture zone and the amount of contacted analyte bindingparticles in the control capture zone; i) determining a correctedanalyte binding particle amount from the amount of analyte bindingparticles in the sample capture zone and the amount of analyte bindingparticles in the control capture zone, wherein the amount of analyte ofinterest in the fluid sample is directly related to the correctedanalyte binding particle amount.
 24. A method for quantitativelymeasuring the amount of an analyte of interest in a fluid sample,comprising: a) providing a solid phase apparatus comprising a membranestrip comprising an application point, a sample capture zone and acontrol capture zone, wherein the sample capture zone is between theapplication point and the control capture zone; b) providing a samplecollection apparatus containing a population of analyte coatedparticles, wherein the analyte coated particles are coated with analyteor an analog of the analyte; c) either i) introducing the fluid sampleinto the sample collection apparatus, producing a mixed fluid sample,and subsequently introducing a buffer into the mixed fluid sample; ii)introducing a buffer into the sample collection apparatus andsubsequently introducing the fluid sample; or iii) forming the fluidsample by introducing a solid into a buffer, and subsequentlyintroducing the fluid sample into the sample collection apparatus,thereby producing a buffered, mixed fluid sample comprising analytecoated particles; d) applying the buffered, mixed fluid sample to theapplication point of the membrane strip; e) maintaining the membranestrip under conditions which allow fluid to transport analyte coatedparticles by capillary action through the strip to and through thesample capture zone, the sample capture zone having a sample capturereagent immobilized thereon; and allow analyte coated particles to bindto the sample capture reagent; f) further maintaining the membrane stripunder conditions which allow the fluid in the sample to transportanalyte coated particles by capillary action through the strip to andthrough the control capture zone, the control capture zone having acontrol capture reagent immobilized thereon; and allow analyte coatedparticles to bind to the control capture reagent; g) further maintainingthe membrane strip under conditions which allow the fluid in the sampleto transport any analyte coated particles not bound to the samplecapture reagent or to the control capture reagent by capillary actionbeyond the control capture zone; h) determining the amount of analytecoated particles in the sample capture zone and the amount of analytecoated particles in the control capture zone; i) determining a correctedanalyte coated particle amount from the amount of analyte coatedparticles in the sample capture zone and the amount of analyte coatedparticles in the control capture zone, wherein the amount of analyte ofinterest in the fluid sample is inversely related to the correctedanalyte coated particle amount.
 25. The method of claim 24, wherein thecorrected analyte coated particle amount is determined as a ratio of theamount of analyte coated particles in the sample capture zone, to theamount of analyte coated particles in the control capture zone.
 26. Themethod of claim 24, wherein the corrected analyte coated particle amountis determined as a ratio of the amount of analyte coated particles inthe sample capture zone, to the sum of the amount of analyte coatedparticles in the control capture zone and the amount of analyte coatedparticles in the sample capture zone.
 27. The method of claim 24,wherein the membrane strip is made of cellulose nitrate or glass fiber.28. The method of claim 24, wherein the particles are latex beads. 29.The method of claim 24, wherein the particles are labeled.
 30. Themethod of claim 29, wherein the label is selected from the groupconsisting of: colorimetric, fluorescent, phosphorescent, luminescent,chemiluminescent, and enzyme-linked molecule.
 31. The method of claim24, wherein the analyte and the sample capture reagent are members of abinding pair, and one member of the binding pair is selected from thegroup consisting of: a spore, a protein, a hormone, an enzyme, aglycoprotein, a peptide, a small molecule, a polysaccharide, a lectin,an antibody, an antibody fragment, a nucleic acid, a drug, a drugconjugate, a toxin, a virus, a virus particle, a portion of a cell wall,a hapten, and a receptor.
 32. The method of claim 24, wherein the samplecapture reagent is selected from the group consisting of: an antibody;an antibody fragment; a hapten; a drug conjugate; and a receptor. 33.The method of claim 32, wherein the sample capture reagent is anantibody.
 34. The method of claim 32, wherein the control capturereagent is an anti-immunoglobulin antibody.
 35. The method of claim 24,wherein the fluid sample is selected from the group consisting of: wholeblood, plasma, serum, urine, cerebrospinal fluid, saliva, semen,vitreous fluid, and synovial fluid.
 36. The method of claim 24, whereinthe fluid sample comprises a suspended solid.
 37. The method of claim36, wherein the solid is selected from the group consisting of: aparticulate sample, a powder sample, a soil sample, and spores.
 38. Themethod of claim 37, wherein the spores comprise spores of Bacillusanthracis.
 39. The method of claim 24, wherein the fluid sample isselected from the group consisting of: water, groundwater, sewage, andwaste water.
 40. The method of claim 24, wherein the analyte of interestis selected from the group consisting of: myoglobin, CK-MB, troponin I,and PSA.
 41. The method of claim 24, wherein in step (d) the mixed fluidsample is applied to the application point through an application pad.42. The method of claim 24, wherein in step (g) the fluid in the sampletransports any analyte coated particles not bound to the sample capturereagent or to the control capture reagent by capillary action beyond thecontrol capture zone into a wicking pad.
 43. The method of claim 24,wherein the sample collection apparatus is selected from the groupconsisting of: a pipette and a pipette tip.
 44. The method of claim 24,wherein the population of analyte coated particles areevaporatively-dried, vacuum-dried or freeze-dried.
 45. A method forquantitatively measuring the amount of an analyte of interest in a fluidsample, comprising: a) providing a solid phase apparatus comprising amembrane strip comprising an application point, a sample capture zoneand a control capture zone, wherein the sample capture zone is betweenthe application point and the control capture zone; b) providing apipette containing a population of analyte coated particles, wherein theanalyte coated particles are coated with analyte or an analog of theanalyte; c) either i) introducing the fluid sample into the pipette,producing a mixed fluid sample, and subsequently introducing a bufferinto the mixed fluid sample; ii) introducing a buffer into the pipetteand subsequently introducing the fluid sample; or iii) forming the fluidsample by introducing a solid into a buffer, and subsequentlyintroducing the fluid sample into the sample collection apparatus,thereby producing a buffered, mixed fluid sample comprising analytecoated particles; d) applying the buffered, mixed fluid sample to theapplication point of the membrane strip; e) maintaining the membranestrip under conditions which allow fluid to transport analyte coatedparticles by capillary action through the strip to and through thesample capture zone, the sample capture zone having a sample capturereagent immobilized thereon; and allow analyte coated particles to bindto the sample capture reagent; f) further maintaining the membrane stripunder conditions which allow the fluid in the sample to transportanalyte coated particles by capillary action through the strip to andthrough the control capture zone, the control capture zone having acontrol capture reagent immobilized thereon; and allow analyte coatedparticles to bind to the control capture reagent; g) further maintainingthe membrane strip under conditions which allow the fluid in the sampleto transport any analyte coated particles not bound to the samplecapture reagent or to the control capture reagent by capillary actionbeyond the control capture zone; h) determining the amount of analytecoated particles in the sample capture zone and the amount of analytecoated particles in the control capture zone; i) determining a correctedanalyte coated particle amount from the amount of analyte coatedparticles in the sample capture zone and the amount of analyte coatedparticles in the control capture zone, wherein the amount of analyte ofinterest in the fluid sample is inversely related to the correctedanalyte coated particle amount. 46-54. (canceled)